Phytochemical properties of Methanol Extract and Antimicrobial study of Less Polar Fractions of Loranthus micranthus (Linn.) parasitic on Alstonia boonei

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Abstract
The leaves of Loranthus micranthus (Linn.) parasitic on Alstonia boonei were extracted with methanol by cold maceration method. The extract was purified further by column chromatographic technique with n-hexane and chloroform respectively. The extract was investigated for phytochemical constituents while the antimicrobial study of the n-hexane and chloroform fractions were evaluated using Escherichia coli and Staphylococcus aureus. The phytochemical study showed tannins, alkaloids, steroids and terpenoids to be moderately present while glycosides, flavonoids and saponins are fairly present. The antimicrobial study of the n-hexane and chloroform fractions showed activities on both bacteria. The minimum inhibitory concentration (MIC) of the n-hexane fraction was 12.42 and 0.49 mg/ml against E. coli and Staph. aureus respectively while the MIC of the chloroform fraction was 23.82 and 0.09 mg/ml against E. coli and Staph. aureus respectively.
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ISSN: 2277- 7695
CODEN Code: PIHNBQ
ZDB-Number: 2663038-2
IC Journal No: 7725
Vol. 2 No. 8 2013
Online Available at www.thepharmajournal.com
THE PHARMA INNOVATION - JOURNAL
Vol. 2 No. 8 2013 www.thepharmajournal.com Page | 83
Phytochemical properties of Methanol Extract and
Antimicrobial study of Less Polar Fractions of Loranthus
micranthus (Linn.) parasitic on Alstonia boonei
John Dike N Ogbonna1*, Benjamin Ebere Ezema2, Chiamaka Peace Obimma2, Matthias Onyebuchi Agbo3
1. Drug Delivery Research Unit, Department of Pharmaceutics
2. Department of Pure and Industrial Chemistry
3. Department of Pharmaceutical and Medicinal Chemistry, University of Nigeria, Nsukka, 410001,
Nigeria.
[Email: johnixus@yahoo.com, Tel: +2348063674303]
The leaves of
Loranthus micranthus
(Linn.) parasitic on
Alstonia boonei
were extracted with methanol by cold
maceration method. The extract was purified further by column chromatographic technique with n-hexane and
chloroform respectively. The extract was investigated for phytochemical constituents while the antimicrobial study
of the n-hexane and chloroform fractions were evaluated using Escherichia coli and Staphylococcus aureus. The
phytochemical study showed tannins, alkaloids, steroids and terpenoids to be moderately present while glycosides,
flavonoids and saponins are fairly present. The antimicrobial study of the n-hexane and chloroform fractions showed
activities on both bacteria. The minimum inhibitory concentration (MIC) of the n-hexane fraction was 12.42 and
0.49 mg/ml against E. coli and Staph. aureus respectively while the MIC of the chloroform fraction was 23.82 and
0.09 mg/ml against E. coli and Staph. aureus respectively.
Keyword: Escherichia coli, Staphylococcus aureus, Loranthus micranthus, Phytochemical analysis.
1. Introduction
In the past, almost all the medicines were from
the plants, the plants being man’s only chemist
for ages. Herbs are staging a comeback, herbal
‘renaissance’ is happening all over the globe and
more and more people are taking note of herbal
therapies to treat various kinds of ailments in
place of mainstream medicine. There is an
increasing demand for medicinal plants and plant
products as alternative to orthodox medicines
especially in developing countries [1]. The need
for more potent, safe and affordable drugs has led
to intensified research into herbal drugs, the result
of which is the introduction of new herbal
preparation for therapeutic uses [2]. The diversity
of biological active substance of plant origin and
their bioactivities are also of current interest
among biomedical research [3]. Researches in
natural alternative medicines exist [4, 5], as there
are increasing evidence that many current drug
therapies simple suppress symptoms and ignore
the underlying disease process, in contrast many
natural products appear to address the cause of
many diseases and yield superior clinical results
Mistletoes which are hemi parasitic plants
growing on different host trees and depend on
their host plant for water and mineral nutrition,
even though they produce their own
carbohydrates through photosynthesis [6, 7]. It is
an obligate semi-parasitic plant of Kola
acuminata, Baphia nitida, Citrus limon, Alstonia
boone, and Pentaclethra macrophylla [8]. The
leaves of mistletoes are traditionally used in
folkloric medicine of Nigeria for the treatment of
diarrhea, epilepsy, hypertension and rheumatism
[2] and the methanol extracts of mistletoes
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parasitic on different host trees have been
reported to have antidiarrhea [9], antidiabetic [10],
antimicrobial [11] and immunomodulatory
activities [12]. Studies have, however, shown that
several factors play important role in the
phytochemical composition and pharmacological
activities of the mistletoe plant, such as: the host,
specie of mistletoe used, season of harvest, etc.
[13, 14, 10, 11, 15]. Earlier studies by these authors on
the crude plant powder and some of its solvent
fractions have established some significant
antibacterial properties by L. micranthus, though
with negligible anti-fungal activity [11, 15, 16].
Fig 1: Diagram of Loranthus micranthus parasitic on Alstonia boonei
Staphylococcus aureus is a Gram-positive
eubacteria found on the surface of human skin
and mucous membrane, other areas of human
contact like air, soil, dust and food products. S.
aureus is an opportunistic pathogen in human and
animals and is one of the most frequent sources
of hospital- and community-acquired infections
[17]. Many isolates of S. aureus have evolved
resistance to both synthetic and traditional
antimicrobial agents posing potential
epidemiological threat outside the hospital; these
isolates can transfer resistant genes to other
potential pathogens [18] and the accumulation of
resistance factors has rendered the bacterium
resistant to a variety of commonly used
antibiotics [19].
This research was to explore the phytochemical
properties and antimicrobial activity of Loranthus
micranthus parasitic on Alstonia boonei on two
different bacteria E. coli and Staph. aureus. This
was because the host plant affects the
phytochemical constituents and hence the bio-
activity of the Loranthus micranthus. Owing to
reported activities, the n-hexane and chloroform
solvent fractions were evaluated for the
antimicrobial activities on these organisms.
2. Materials and Methods
2.1 Materials
Methanol (Riedel de Hain, Germany), n-hexane
(Riedel-de Haen, United Kingdom), chloroform
(Sigma-Aldrich®, Germany), Thomas-Wiley
Laboratory Mill, (Model 4) and distilled water
(Lion Water, University of Nigeria, Nsukka)
2.1.1 Plant Material
The leaves of L. micranthus parasitic on Alstonia
boonei were collected in January 2011 from
Enugu-Ezike, Enugu State and were identified by
Mr. A. O Ozioko, of Bio-resources Development
and Conservation Programme (BDCP), Nsukka
and a voucher specimen (LM1610) was deposited
at the herbarium of the Institute. All other
reagents were of analytical grade and were used
without further purification.
The Pharma Innovation - Journal
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2.2 Methods
2.2.1 Preparation of the plant material
The leaves of L. micranthus parasitic on Alstonia
boonei were air-dried and extracted according to
the procedure by Agbo et al., [7] with slight
modification. Briefly the leaves were pulverized
and the powder (500 g) extracted by cold
maceration method in 3.0 L methanol at room
temperature for 48 h with continuous agitation.
The extract was concentrated in vacuo at 40 oC
and stored in the refrigerator till when used.
2.2.2 Purification of the methanol extracts
using column chromatographic technique
Ten gram (10.0 g) of the dried methanol extract
was purified further using column
chromatographic method. The extract was mixed
with silica gel 60 and dissolved in methanol. The
mixture was dried and loaded unto glass column
(150×1.5 cm, ID) which has been packed to two-
third the length with 200 g slurry of silica gel (20-
230 mesh). The column was eluted with 2.5 L of
n-hexane and 2.5 L of chloroform. These
fractions were concentrated in vacuo to obtain the
dry solvent fractions.
2.2.3 Phytochemical screening of the methanol
extract
Phytochemical analysis of the extract was
performed using standard method [20, 21].
2.2.4 Determination of antimicrobial
susceptibility
The antimicrobial susceptibility studies of the
solvent fractions (n-hexane and chloroform) were
carried out using the paper disc diffusion
technique previously described by
Cheebrough[22]. The stock solution of the n-
hexane fraction was prepared as described below.
Standard concentrations of the fraction was
introduced into different 6 mm diameter paper
discs with micropipette and preserved aseptically.
The densities of the microorganisms were
adjusted as per Mac Farland 0.5 standard and
microorganisms were grown in nutrient broth for
18 h, and transferred to Muellar Agar (MHA).
The n-hexane fraction impregnated disc was
introduced into the Muellar Hinton Agar already
inoculated with the test microorganisms. The
plates were incubated at 37 oC for 24 h and zones
of inhibition of the triplicate analyses were
measured and the mean IZD determined. The
same procedure was repeated for the chloroform
fraction.
2.2.5 Determination of MIC of the different
fractions
The MIC was determined using agar diffusion
method. A stock was prepared by dissolving 100
mg of the fractions in 2 mL of Tween 80 or
DMSO to get 50 mg/mL. A six two fold serial
dilution of the stock (25, 12.5, 6.25, 3.13, 1.56
and 0.78 mg/mL were prepared. 24 h MacFarland
standard of E. coli and Staph. aureus were
prepared with sterile normal saline and 0.1 mL of
each organism was smeared over the face of a
sterile nutrient agar plate and 20 mL of the
MacFarland standard was added and swirled for
uniform distribution. Sterile cork borer was used
to bore hole on the nutrient agar plate. This was
allowed to stay on the bench for pre-diffusion
time of 1 h and later incubated for 24 h. The zone
of inhibition was measured and the mean
inhibition zone diameter determined.
2.3 Statistical analysis
The results were analyzed using descriptive
statistical method according to Woodson [23].
3. Results
The yield was assessed to know which of the
solvent will be adopted in fractionating the L.
micranthus. The yield of the fractions is shown in
Table 1. The methanol extract had the highest
yield while the chloroform extract had the lowest
yield.
3.1 The Phytochemical study
Phytochemical screening of the methanol extract
of the leafy twigs of L. micranthus parasitic on
Alstonia boonei led to the identification of many
bioactive constituents as shown on Table 2.
The use of herbal remedies in antimicrobial
treatment is a common practice in many countries
of the world including Nigeria. The potential
inhibitory effects of n-hexane and chloroform
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fractions of the methanol extract of Loranthus
micranthus was evaluated on selected human
pathogens- E. coli and Staph. aureus.
Table 1: Percentage Yield of Extract and Fractions.
Extract/Fractions Colour Mass (g) Yield (%)
Methanol extract Greenish black 10.00 2.00
n-hexane Yellow 2.50 0.51
Chloroform Greenish 0.60 0.12
Table 2: Phytochemical Studies of the Plant Extract
Relative
abundance
Alkaloids
++
Glycosides
+
Saponins
+
Steroids
++
Terpenoids
++
Tannins
++
Flavonoids
+
KEY: + Low in concentration, ++ Moderate in concentration
Table 3: Various concentrations (mg/ml) of n-hexane fraction of Loranthus micranthus leaves against
Staph. aureus and E. coli
Staph. aureus E. coli
Conc. (mg/ml) Log Conc.
Mean IZD
(mm) Mean IZD2 (mm²)
Mean IZD
(mm)
Mean IZD
2
(mm²)
50.00
1.6990
6.02 ± 0.15
36.24
2.02 ± 0.02
4.08
25.00
1.3979
5.00 ± 0.00
25.00
2.00 ± 0.00
4.00
12.50
1.0969
4.15 ± 0.22
17.22
+
+
6.25
0.7959
4.12 ± 0.20
16.97
+
+
3.13
0.4949
4.00 ± 0.00
16.00
+
+
1.56
0.1938
3.00 ± 0.00
9.00
+
+
0.78
-
0.1072
2.50 ±0.20
6.25
+
+
Key: + no activity or growth not inhibited.
Table 4: Various concentrations (mg/ml) of chloroform fraction of Loranthus micranthus leaves against
Staph. aureus and E. coli
Staph. aureus E. coli
Conc. (mg/ml) Log Conc. Mean IZD (mm)
Mean IZD
2
(mm²) Mean IZD (mm)
Mean IZD
2
(mm²)
50.00
1.6990
4.00
±
0.00
16.00
1.00 ±
0.00
1.00
25.00
1.3979
3.13
± 0.13
9.80
3.00 ±
0.00
9.00
12.50
1.0969
3.10
±
0.10
9.61
+
+
6.25
0.7959
3.07
± 0.02
9.42
+
+
3.13
0.4949
3.02
±
0.02
9.12
+
+
1.56
0.1938
3.00
±
0.00
9.00
+
+
0.78
-
0.1072
3.00
±
0.00
9.00
+
+
Key: + no activity or growth not inhibited.
4. Discussion
The in vitro results of this study showed that the
n-hexane and chloroform fractions of the plant
were active against the test organisms.
Tables 1 and 2 showed that the fractions were
more active against Staph. aureus being a Gram
positive bacteria than E. coli. The MICs of the n-
hexane and chloroform fraction were 0.49 mg/ml
The Pharma Innovation - Journal
Vol. 2 No. 8 2013 www.thepharmajournal.com Page | 87
and 0.09 mg/ml respectively against Staph.
aureus and 12.42 and 23.82 mg/ml respectively
against E. coli.
Fig 2: MICs of n-hexane and chloroform fractions of L.
micranthus on E. coli and Staph. aureus
This showed that the fractions are more suitable
for the treatment of any infection caused by
Staph. aureus than E. coli. The reason for this can
be drawn from the antimicrobial activity of
amoxicillin used as a reference drug against both
bacteria which has a MIC of 0.0000254 mg/ml
and 0.0145 mg/ml respectively. Amoxicillin
(penicillin) is more active against gram positive
bacteria because they inhibit the growth of
peptidoglycan crosslinks in the bacteria cell wall
thereby weakening the cell wall; this imbalance is
responsible for the rapid killing action of
amoxicillin. This gives the mode of action
thereby explaining the greater activity of the
fractions against S. aureus.
The result of the phytochemical screening of the
methanol extract of Loranthus micranthus
showed the presence of different phytochemical
constituents in several degrees of abundance. The
presence of tannins, saponins and alkaloids
enhanced the antimicrobial activity thereby
increasing its broad spectrum of activity [7]. An
analysis of the IZD’s and phytochemical results
suggested that the antibacterial activity observed
in L. micranthus might have arisen as a result of a
number of the phyto-constituents present in the
plant. This followed from the results which
clearly showed that no singular
fraction/constituent could be said to be solely
responsible for the antibacterial action of the
plant. Among these constituents, however,
tannins, flavonoids, terpenoids and alkaloids
appear to have the greatest impact on the activity
under study [10]. Metabolites like flavonoids and
terpenoids are known to be synthesized by plants
in response to microbial infection, and thus have
been found in vitro to be effective antimicrobial
substances against a wide array of
microorganisms) [24].
5. Conclusion
The solvent fractions have displayed
antimicrobial activity and therefore justify the
ethno medicinal uses of mistletoe in the treatment
of bacteria infections. These solvent fractions are
quite active but below those of standard
antibiotics (Amoxicillin). Therefore, it is
recommended that further investigations should
be done on the chemical nature of the active
components of the plant. This knowledge will
help pharmaceutical chemists to alter the
structure of these components in order to enhance
their lethality on the test organisms.
6. Conflict of interest
The authors wish to declare no conflict of
interest.
7. Acknowledgments
The authors wish to thank Mr. Alfred Ozioko
(Bioresources Conservation and Development
Program (BDCP), Nsukka, Nigeria) for
identifying the plant material.
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